1. Field of Invention
The present invention relates to a method of making rapid, definitive identification of human leukocytic antigens (HLA) by DNA analysis and the device thereof. The present invention also relates to a method of making rapid, definitive identification of a human or any organism by DNA analysis and the device thereof.
2. Description of Related Arts
(a) Identification of Human Leukocytic Antigens (HLA)
Accurate HLA typing is essential for matching donor and recipient in organ or marrow transplantation (4) to prevent the development of acute graft-versus-host disease (GVHD) . This is generally accomplished by standard serological typing (2) . Recent studies have demonstrated that DNA genotyping can provide more accurate and definitive result (7, 9, 8). Results of HLA-DQ, DR and DP genotyping provided data for accurate matching which is necessary in selecting potential organ donors (3). HLA genotyping using sequence-specific primers polymerase chain reaction (SSP-PCR) amplifications has been previously reported. However, due to highly polymorphic nature of HLA-DQ, DR and DP loci, the number of SSP required will be in the hundreds, and, therefore, exceed the limit of multiplex-PCR for efficient PCR amplification. To ensure the results of HLA genotyping have practical clinical application, multiple of 50 to 100 of separate PCR reactions have to be setup. A kit that is available on the market today includes an amplification process of carrying out a 96 PCR separate reactions, then followed by analysis on gel electrophoresis size separation. This is not only time consuming and costly but prone to error due to the complexity of the reaction setup and sizing uncertainty of the gel electrophoresis. Thus, DNA sequencing is still considered the method of choice for accurate genotyping of the HLA cluster. Unfortunately, because of the existence of highly homologous sequence of pseudogene(s) that may be co-amplified during the polymerase chain reaction (PCR) amplification process, accurate genotyping by DNA sequencing alone may prove more difficult and costly. U.S. Pat. Nos. 5,471,547 and 6,020,187, issued to J .W. O. Tam, disclose a fast annealing process that uses a very inexpensive device for accurate mutation detection, genotyping and fingerprinting analysis. The present invention discloses a method of rapidly analyzing HLA loci of DP, DR and DQ beta sequences by ASO oligo-probes using an improved flow-through format.
(b) DNA Fingerprinting for Rapid Identification of Human Beings and Organisms
DNA fingerprinting by restriction fragment length polymorphism (RFLP) was first introduced in 1985 (12) for human identification, and was subsequently applied to identification of other organisms. In practical applications, DNA fingerprinting has been widely accepted as the best forensic tool for identification of suspects in criminal cases, for paternity disputes and for establishing or verifying the identity of a person. The time consuming RFLP method has been gradually replaced by high throughput automated processes. Using PCR amplification for analyzing the number of short tandem repeat (STR), first discovered in 1991 (11), from 10, 16, 18 or more loci in the human genome, single cell identification is now possible. However, both STR and/or variable number of tandem repeats (VNTR) are relatively expensive because these methods require the use of sophisticated equipment, and labor intensive and time consuming process like the Southern blotting hybridization. Sporadic mutations (10) may reduce the accuracy and the power for definitive identification. Furthermore, STR data suggested that the frequency of mutation, particularly in cancer patients, is not uncommon. Hence, new alternative method is needed. Single nucleotide polymorphism (SNP) genotyping provides greater discriminating power by selecting an appropriate number of SNPs at unlink loci, and the mutation frequency in each locus (site) is lower than VNTR or STR systems for forensic or individual personal identification. This invention presents a method of making rapid, definitive biometric identification of an individual, such as a human being, animal, plant or any organism, using SNP genotyping.